Amphiregulin - a new indicator for zygote vitrification

Abstract Study question Does the concentration of amphiregulin in follicular fluid affect survival rate and embryo quality after the vitrification process? Summary answer The concentration of amphiregulin affects development potential and embryo quality after zygote vitrification and can be used as a predictive biomarker. What is known already Epidermal growth factor receptors and its ligands, such as amphiregulin(AR), which are expressed in female reproductive tissues, have been shown to regulate various important reproductive functions. Amphiregulin concentration in follicular fluid is correlated with human oocyte developmental competence and IVF outcomes. Unlike other countries, Germany has a specific Embryo Protection Act (EPA), where the choice of embryos that have the highest potential for successful implantation must be made at the pronuclear stage(PN); in addition, all supernumerary fertilized oocytes are cryopreserved on the zygote stage. Due to EPA, the determination of potential biomarkers is key for successful IVF outcomes in Germany. Study design, size, duration This study consisted a total of 43 vitrified 2PN stage zygotes from 19 patients who underwent ICSI at UKS Homburg, (Germany) between April 2020 and November 2020. For the study purpose, follicles were aspirated separately, and the follicular fluid (FF) was centrifuged for 5 min at 1,500×g, after that supernatant was removed and stored at -20. Zygote vitrification was performed according to company guidelines (RapidVit Omni, Vitrolife). Participants/materials, setting, methods Concentrations of AR in FF were determined by using a commercially available sandwich ELISA kit (DuoSet Kit; R&D Systems Inc., Minneapolis) in accordance with the manufacturer’s instructions. Good embryo quality on day 3 was defined as a 7-9 cell stage embryo with less than 25% of fragmentation and with equally sized blastomeres. Between-group comparisons were tested using a t-test. Logistic regression analysis was used to determine the influence of amphiregulin on vitrification outcomes. Main results and the role of chance The mean age of all patients was 31.6 (±3.5) years. Patients included in the study were stimulated according to a gonadotropin-releasing hormone (GnRH) antagonist protocol. All zygotes were frozen 20h after injection. Out of the 43 frozen zygotes, 38 (88.3%) survived the thawing process and continued development. Obtained results showed that concentrations of amphiregulin were significantly higher in FF where zygote continued the development [(95.88±37.02 ng/mL vs 79.19±9.92 ng/mL) respectively; p < 0.05]. In addition, 26 (68.4%) vitrified zygotes developed into good-quality embryos, while 17 (31.6%) embryos had poor quality. Data comparisons between good and poor-quality embryos indicated that good-quality embryos came from follicles where amphiregulin concentration was significantly higher [(113.52 ±30.36 ng/mL. vs 63.98 ±1.93 ng/mL) respectively; p < 0.0001]. What is more, logistic regression analysis confirmed the positive effect of amphiregulin concentration on embryo quality (p < 0.01). Limitations, reasons for caution The limitation of the presented study was a relatively small patient cohort. Although the pregnancy rate in the presented study was 36.8 % single embryo transfer was not performed, therefore further research should link amphiregulin concentrations with blastocyst quality and pregnancy rate as well. Wider implications of the findings The literature already confirmed a lower development potential of vitrified zygotes compared to cleaved embryos or blastocyst. Therefore, obtained findings might be helpful for the success of the vitrification process in Germany. Additionally, our findings can be a basis for the future study of amphiregulin as a biomarker. Trial registration number not applicable